Oncolytic HSV vectors (oHSV) offer considerable promise in the treatment of Glioblastoma Multiforme (GBM). In previous studies we designed a new base vector (KGN-4:T124) that is blocked for replication in normal brain tissue by brain-specific cellular miR-124 targeting of ICP4 expression. This base vector was further retargeted (KGNE-4:T124) to achieve selective infection of GBM cells expressing EGFR/EGFRvIII. Arming of this vector with a matrix metalloproteinase (KGNE-4:T124-MMP9) enhanced vector spread and oncolysis in xenogeneic animal models. However, virolysis alone is unlikely to achieve complete elimination of tumor cells that can cause recurrence. Effective elimination of all tumor cells will require the induction of innate and adaptive anti-tumor immunity. Unfortunately, GBM down-regulates the cellular machinery needed to sustain activation of immune cells including NK, macrophages and cytotoxic T cells. Therefore we propose to test the hypothesis that GBM treatment with our oHSV base vectors, KGN-4:T124 and KGNE-4:T124, can be enhanced through vector arming with immunomodulatory genes that inhibit tumor progression and induce innate and adaptive anti-tumor immune responses. To this end, we will initially exploit a tumor cell line (BAGL1) derived from a GBM tumor induced in BALB/c mice by Sleeping Beauty transposition of genes encoding luciferase, anti-p53 shRNA, N-RasV12 and human EGFRvIII. These experiments will be extended to a genetically-induced GBM model developed by Dr. Eric Holland that avoids tumor cell injection. Our aims will seek to counteract three aspects of immune evasion in GBM: (i) ADAM17-mediated shedding of NK cell activators and reduced NK function, (ii) expression of the immune checkpoint molecules that lead to NK and T cell exhaustion, and (iii) the presence of M2 macrophages that contribute to an immunosuppressive tumor microenvironment (TME). In Aim 1, we will test the hypothesis that our unarmed base vectors, collectively referred to as KGN(E)-4:T124, will provide an effective GBM treatment in immune competent mice. We will determine the impact of vector dosing on intratumoral vector distribution, TME composition and animal survival, and assess the value of EGFR targeting. We will also examine the effect of prior HSV immunization on treatment outcomes. In Aim 2, we will test the hypothesis that KGN(E)- 4:T124 derivatives armed with TIMP-3 (ADAM17 inhibitor/VEGF-R2 antagonist) will improve therapy. We will evaluate the impact of TIMP-3 expression on NK cell activation, tumor spread and animal survival. In Aim 3, we will test the hypothesis that vectors armed with single chain antibody (scFv) checkpoint inhibitors (anti-PD-L1 or anti-CTLA4), alone or in combination with depletion of the immunosuppressive (M2) TAM population using BLZ945-mediated inhibition of colony-stimulating factor-1 receptor (CSF-1R), will produce effective anti-tumor immunity, particularly in HSV immune mice. Anti-tumor immunity will be established using a bi-lateral tumor model and confirmed by selective antibody-mediated T cell depletion.